Fluorescence In Situ Hybridization Assay Research Articles

Determination of True ERBB2 Gene Amplification in Breast Cancer by Quantitative PCR Using a Reference and a Novel Control Gene.

Human epidermal growth factor receptor 2 (ERBB2/HER2) is amplified and overexpressed in 20% to 25% of breast carcinomas, correlates with poor outcome, and is an indication for treatment with trastuzumab. Accurate assessment of ERBB2 status is crucial for proper prognosis and to offer appropriate treatment for patients. ERBB2 status is generally determined by immunohistochemistry or fluorescence in situ hybridization (FISH), and sporadically by quantitative real-time polymerase chain reaction (PCR). We developed a new algorithm, termed quantitative PCR algorithm (QPA) score, and compared its performance with the gold standard FISH assay. The QPA is a computation of the relative number of copies of the ERBB2 gene with respect to a nonstandard, short-arm centromeric sequence on chromosome 17, and referenced to a single-copy gene, RPP30. This provides a more reliable determination of ERBB2 amplification, reducing the false polysomy 17 error. A total of 69 breast carcinoma samples were tested for quantitative real-time PCR and FISH, and the degree of concordance was analyzed. Sixty-two cases were in agreement between the 2 methods, and the contingency study assigned a κ value of 0.729 for their correlation. A receiver operating characteristic analysis was used to determine the optimal cut-off point for ERBB2 amplification, which was estimated at a QPA=1.53 (sensitivity=0.863; specificity=0.944). Our data conclude that the QPA is able to determine ERBB2 gene status with high accuracy, while also overcoming the limitations of conventional techniques and providing better cost-effectiveness.

In prostate cancer needle biopsies, detections of PTEN loss by fluorescence in situ hybridization (FISH) and by immunohistochemistry (IHC) are concordant and show consistent association with upgrading.

The prognostic value of phosphatase and tensin homolog (PTEN) loss in prostate cancer has primarily been evaluated by either fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). Previously, we found that PTEN loss by IHC was associated with increased risk of upgrading from biopsy (Gleason 3 + 3) to prostatectomy (Gleason 7+). Now, using an evaluable subset of 111 patients with adjacent biopsy sections, we analyzed the association between PTEN deletion in cancer and the odds of upgrading by a highly sensitive and specific four-color FISH assay. We also compared the concordance of PTEN loss by IHC and PTEN deletion by FISH. PTEN deletion was found in 27% (12/45) of upgraded cases compared with 11% (7/66) of controls (P = 0.03). Cancers with PTEN deletions were more likely to be upgraded than those without deletions (adjusting for age odds ratio = 3.40, 95% confidence interval 1.14-10.11). With respect to concordance, of 93 biopsies with PTEN protein detected by IHC, 89 (96%) had no PTEN deletion by FISH, and of 18 biopsies without PTEN protein by IHC, 15 had homozygous or hemizygous PTEN deletion by FISH. Only 4 biopsies of the 93 (4%) with PTEN protein intact had PTEN deletion by FISH. When the regions of uncertainty in these biopsies were systematically studied by FISH, intra-tumoral variation of PTEN deletion was found, which could account for variation in immunoreactivity. Thus, FISH provides a different approach to determining PTEN loss when IHC is uncertain. Both FISH and IHC are concordant, showing consistent positive associations between PTEN loss and upgrading.

Epithelioid hemangioendotheliomas with TFE3 gene translocations are compossible with CAMTA1 gene rearrangements.

Epithelioid hemangioendotheliomas (EHEs) are vascular tumors of intermediate malignancy that can undergo high-grade malignant transformations. EHEs have been characterized by tumor-specific WW domain-containing transcription regulator 1(WWTR1)-calmodulin-binding transcription activator 1 (CAMTA1) translocations, and recently, a novel Yes-associated protein 1 (YAP1)-transcription factor E3 (TFE3) gene fusion was identified in EHEs. In this study, we examined the expression levels of TFE3 and CAMTA1 via immunohistochemical staining and identified chromosomal alterations using fluorescence in situ hybridization (FISH) assays and RT-PCR tests. Although all of the EHEs were CAMTA1-positive in immunohistochemical staining, only five out of 18 EHEs (27.78%) positively expressed nuclear TFE3. The five TFE3-positive EHEs exhibited TFE3 gene break-apart in FISH assays. YAP1-TFE3 gene fusions were confirmed by RT-PCR. Interestingly, we observed CAMTA1 gene break-apart in all of the five TFE3-positive EHEs via FISH assays, and four out of the five TFE3-positive EHEs exhibited WWTR1-CAMTA1 gene fusions via RT-PCR. These results indicate that these two chromosomal alterations are not mutually exclusive but compossible in EHEs. Finally, primary tumor sites in TFE3-positive EHEs consistently contained single masses (P = 0.0359) with larger sizes (P = 0.0550) compared to TFE3-negative EHEs. Similar to previous reports, we observed well-formed vessels more frequently in TFE3-positive EHEs than in TFE3-negative EHEs (P = 0.0441). In addition, TFE3-positive EHEs tended to more frequently demonstrate high-grade nuclear atypia (P = 0.0654) and hypercellularity (P=0.0987) than TFE3-negative EHEs. Thus, we have now established two clinically distinct subgroups of EHEs: TFE3-positive and TFE3-negative EHEs.

MLL translocation in two castration-resistant prostate cancer (CRPC) patients.

186 Background: The mixed-lineage leukemia (MLL) protein is a histone methyltransferase that regulates multiple genetic elements. Chromosomal rearrangements of the MLL gene result in expression of MLL-fusion proteins that occur in a subset of acute leukemias and are associated with poor prognosis. The MLL protein complex has been shown to interact with the androgen receptor via the MLL-menin subunit and, in model systems, MLL-menin inhibition blocks CRPC growth. We describe 2 cases of metastatic CRPC with a translocation in the MLL gene. Methods: In our institution, fluorescence in situ hybridization (FISH) testing of bone marrow specimens is routinely performed using the MLL Breakapart probe from Cytocell, Ltd (Cambridge, UK). The probe consisted of an 87 kb segment labeled in red that covered a region telomeric to the MLL locus and a 168 kb segment labeled in green centromeric to MLL. The FISH assay was performed according to manufacturer’s instructions. Results: MLL translocation was found coincidentally in 2 patients. The first was a 50 year old male with CRPC who had progressed on abiraterone and multiple chemotherapy regimens. A bone marrow biopsy was done to evaluate pancytopenia and pathology revealed metastatic prostate cancer marrow infiltration, without any evidence of leukemia or myelodysplasia. FISH studies revealed a rearrangement of the MLL locus using the MLL Breakapart probe. The second patient was a 77 year old male with metastatic CRPC who had also progressed through multiple hormonal and chemotherapy regimens. Bone marrow biopsy was done to evaluate thrombocytopenia and pathology revealed metastatic prostate cancer occupying nearly 100% of the marrow. Cytogenetics revealed complex karyotype and FISH was positive for MLL gene rearrangement by the same assay. There was no evidence of leukemia or myelodysplasia in either case. Conclusions: Translocation of the MLL gene is well documented in leukemia but has not been described in CRPC. Additional studies are warranted regarding the frequency and importance of this potential therapeutic target.

Alveolar soft part sarcoma: a clinicopathologic analysis of 48 cases

To study the clinicopathologic features and differential diagnosis of alveolar soft part sarcoma (ASPS). The clinical data and pathologic features of 48 cases of ASPS were evaluated. Immunohistochemical study, PAS staining and fluorescence in-situ hybridization (FISH) were carried out in selected examples. Relevant literature was reviewed. Amongst the 48 cases studied, there were 17 males and 31 females, with male-to-female ratio of 1.0∶1.8. The age of patients ranged from 2 to 60 years (median=26 years). The tumor was most commonly located in deep soft tissue, especially that of lower extremities. Histologically, the tumor cells were arranged in alveolar or solid patterns and separated by sinusoidal vessels. They were large and contained abundant eosinophilic granules or crystals in cytoplasm. The nuclei were round to polygonal and vesicular, often with prominent nucleoli. Intravascular tumor extension was common. Some cases showed necrosis, hemorrhage and cystic changes. Immunohistochemical study showed that the tumor cells were positive for TFE3 (100%, 33/33). FISH assay was carried out in 4 cases and all of them had TFE3-ASPL gene fusion. ASPS is a rare malignant neoplasm, often occurs in young patients. TFE3 is a useful immunohistochemical marker for diagnosis. The diagnosis is further confirmed by other markers.

Three Steps to the Diagnosis of Adult Ph-like ALL

Introduction: Philadelphia chromosome-likeacute lymphoblasic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to BCR-ABL-positive ALL and is associated with particularly poor prognosis. Aberrant gene-expression, point mutations or fusion translocations cause activation of either the ABL1 or JAK signaling pathways. The diagnosis of Ph-like ALL is challenging but of vital importance as it is likely to respond to targeted therapy with ABL or JAK inhibitors.Aims: To develop a robust and efficient stepwise diagnostic work-up to identify Ph-like ALL in routine diagnostics.Patients and Methods: We studied 132 adult precursor B-ALL patients (pts) lacking the recurrent aberrations BCR-ABL1, MLL-AF4 and E2A-PBX1 (age 18-85 years, median 54 years). Precursor B-ALL was diagnosed by immunophenotyping in all cases. The following methods were performed in parallel: Microarray gene-expression profiling (GEP) according to Roberts et al, NEJM 2014; fluorescence in situ hybridization (FISH) with customized probes for ABL2, TSLP, CSF1R, PDGFRB, PTK2B, JAK2, ETV6, IGH, NTRK3, EPOR, TYK2, and IL2RB; and RT-PCR for detection of ABL and JAK pathway activating fusions involving the genes ABL1, ABL2, CSF1R, PDGFRB and JAK2; quantitative real-time PCR for evaluation of CRLF2-expression, fragment analysis for detection of IKZF1 deletions; next generation sequencing for mutations in JAK1, JAK2, JAK3, KRAS, and NRAS.Results: Based on GEP, we identified 13/132 adult precursor B-ALL pts (10%) as Ph-like ALL. As an alternative approach, we applied a combination of FISH, RT-PCR, fragment analysis and next generation sequencing for diagnosis of Ph-like ALL and simultaneous identification of therapy sensitive targets. Quantitative real time PCR identified 11/132 cases with CRLF2 overexpression (8%), of which 9 cases were also classified as Ph-like ALL according to GEP. 7/11 (64%) cases with elevated CRLF2 expression harbored IGH-CRLF2 rearrangement detected by our FISH assay. Additionally, RT-PCR and FISH analysis identified 4 pts with rearrangements involving TKI sensitive genes ABL1 (SNX2-ABL1; n=1), and PDGFRB (EBF1-PDGFRB; n=3). Rearrangements of EPOR and JAK2 were not detected. Taken together, the combination of quantitative RT-PCR, FISH and fusion specific RT-PCR identified 15 cases (12%) compared to 13 cases (10%) identified by GEP with Ph-like ALL in a cohort of 132 preselected adult precursor B-ALL cases: 13 pts were assigned to the Ph-like subset by both approaches, while no patient was identified by GEP only and two were identified by the alternative approach only.Mutations in JAK2 were detected in 8/15 Ph-like ALL cases, all of which were characterized by CRLF2 rearrangements and/or overexpression of CRLF2. In comparison, none of the 117/132 non Ph-like ALL cases harbored JAK2 mutations. NRAS mutations were found in two pts with CRLF2 rearrangement. Mutations in JAK1, JAK3 and KRAS were not detected. Furthermore, we found a high frequency of IKZF1 deletions in Ph-like ALL pts (n=12/16; 75%) compared to 19% (22/117) in the remaining 117 cases with non Ph-like ALL (p<0.001), which is in line with previous published data.Regarding age, 5/15 Ph-like pts were young adults (age: 31-39 years), 3/15 pts adults (age: 40-55 years) and 7/15 pts were older adults (age: 56-76 years), indicating an incidence of Ph-like ALL of 8% in adults age ≥40 years.Conclusions: In adult precursor B-ALL lacking the recurrent aberrations BCR-ABL1, MLL-AF4 and E2A-PBX1 Ph-like ALL was identified in 10% of pts based on GEP and in 12% by an alternative approach based on RT-PCR and FISH, respectively. As GEP per se is challenging in routine diagnostics and does not provide therapeutic information regarding potential susceptibility to ABL or JAK inhibitors, we postulate the following diagnostic algorithm to identify Ph-like ALL: 1.Analysis of CRLF2 expression.2.FISH targeting ABL and JAK pathway activating fusions involving the genes ABL1, ABL2, CSF1R, PDGFRB and JAK2.3.Fusion specific RT-PCR for the identification of the respective ABL and JAK fusion partner and sensitive MRD monitoring.We show that the alternative approach is efficient for identification of Ph-like ALL and is feasible for routine diagnostics. DisclosuresFasan:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schlenther:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Clinical Validation of a Novel Commercial Reverse Transcription–Quantitative Polymerase Chain Reaction Screening Assay for Detection of ALK Translocations and Amplifications in Non–Small Cell Lung Carcinomas

-EGFR mutations and anaplastic lymphoma kinase (ALK) translocations have significant biologic and therapeutic implications in lung cancers, particularly lung adenocarcinomas. ALK translocations are less frequent compared with EGFR mutations; interestingly, these two abnormalities are most commonly mutually exclusive. The 2013 College of American Pathologists/Association for Molecular Pathology/International Association for the Study of Lung Cancer molecular testing guideline for lung cancers recommend a testing algorithm in which detection of ALK translocations using fluorescence in situ hybridization (FISH) is to be performed following testing for EGFR mutations. Such an algorithm is cost-effective but potentially slows down turnaround time; and as a secondary test, ALK FISH assay may not be completed because it requires the use of additional tissue, and the small biopsies or cytology specimens may have been exhausted in the extraction of nucleic acid for EGFR mutation screening. -To provide efficient testing of both EGFR and ALK genetic alterations in small biopsies and cytology specimens. -We validated a highly sensitive ALK reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay as a screening tool for ALK translocations and amplifications. -We performed a retrospective review of cases previously tested by FISH and found that all FISH ALK translocation-positive specimens were RT-qPCR positive, and all FISH ALK translocation-negative cases were RT-qPCR negative (the sensitivity and specificity of the ALK RT-qPCR assay were 100%). -This assay allows rapid identification of ALK alterations, can be performed in conjunction with EGFR testing, and does not require use of valuable additional tumor tissue.

Epidermal Growth Factor (EGFR) copy number aberrations in esophageal and gastro-esophageal junctional carcinoma.

BackgroundClinical trials of agents targeting epidermal growth factor receptor (EGFR) in esophageal carcinoma (EC) have indicated a minority subgroup responsive to anti-EGFR therapies. Other investigations suggest increases in EGFR copy number are associated with poor prognosis in EC, but have used a variety of different techniques and tested numbers remain small. A validated assay for EGFR copy number in EC is needed, to allow investigation of EGFR copy number gain as a predictive biomarker for the anti-EGFR responsive subgroup of patients. We developed a scoring system in EC based upon established systems for EGFR fluorescence in-situ hybridisation (FISH) in lung cancer, and applied this in a series of 160 UK patients with advanced EC.ResultsDual colour FISH on formalin fixed paraffin embedded (FFPE) biopsies were scored independently by two operators as: disomy (score = 1), low trisomy (score = 2), high trisomy (score = 3), low polysomy (score = 4), high polysomy (score = 5) and amplification (score = 6). EGFR FISH positive cases (scores 5 and 6) were found in 32/160 (20 %) tumours, with high polysomy in 22 (13.8 %) and amplification in 10 (6.3 %). Two independent operator scores for FISH positivity were 100 % concordant. EGFR FISH positive status was not associated with clinic-pathological features. EGFR amplification was associated with worse survival (HR = 2.64, 95 % CI 1.04 to 6.71, p = 0.03).ConclusionOur FISH scoring system for EGFR in advanced EC identifies a significant subgroup (20.0 %) of FISH positive patients. EGFR amplification, which is found in 6.3 %, is associated with poor survival. It is not known if there is a role for EGFR targeted treatment in this subgroup of patients, however we are now utilising this EGFR FISH assay and scoring system in biopsies from clinical trials utilising anti-EGFR targeted therapies.

A standardized protocol for detection of ALK protein expression and gene fusion in lung adenocarcinoma cytologic specimens

The aim of this study was to establish a standardized protocol for detection of ALK protein expression and gene fusion in cytologic specimens. Lung adenocarcinoma cytologic specimens were collected from seven hospitals in Beijing city. A detection protocol for ALK protein expression and gene fusion was designed according to the results of comparative experiment. Ventana immunohistochemical (IHC) ALK(D5F3) detecting ALK protein expression was performed in 203 prepared formalin-fixed paraffin-embedded (FFPE) cell blocks. ALK gene fusion in 98 EGFR gene wild type cytologic specimens and in 4 bronchoalveolar lavage fluid (BL) samples was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). ALK gene fusion in the Ventana IHC ALK (D5F3) positive samples was further tested by fluorescence in situ hybridization (FISH). Six patients with ALK IHC-positive result were followed up to analyze the responses of crizotinib therapy. Comparative experiments: (1) Comparison of the results of 4% neutral buffered formalin fixed for different time (24 h, 48 h, 72 h) on the Ventana IHC ALK (D5F3) staining was conducted in two cases of IHC ALK positive FFPE cell blocks; (2) Comparing qRT-PCR results for ALK fusion in samples from FFPE cell blocks and cytospin prepared slides in 10 cases of lung adenocarcinoma cytologic specimens. Among the specimens examined using the standardized protocol recommended by this study, 229 cases of cytologic specimens met the diagnostic criteria of lung adenocarcinoma. Among them, 207 cases obtained ALK gene test results (by at least one method), with an ALK test ratio of 90.4% (207/229). FFPE cell blocks were successfully prepared in 203 cases, Ventana IHC ALK (D5F3) were successfully performed in all the 203 FFPE cell blocks (100%), and the ALK protein positive detection rate was 10.3% (21/203). ALK fusion was tested in 98 FFPE cytologic samples of EGFR wild types by qRT-PCR, and 96 out of 98 (97.96%) cytologic samples were successfully performed.18 out of 19 IHC ALK-positive cases were verified to be of ALK fusion status by qRT-PCR. The concordance rate was 94.7% (Kappa=0.967, P<0.001) between Ventana IHC ALK (D5F3) and qRT-PCR, and the sensitivity of the Ventana IHC ALK (D5F3) assay compared with qRT-PCR was 100% and the specificity was 98.7%. FISH assay was used to verify the positive cases detected by Ventana IHC ALK (D5F3) staining. Two cases of low tumor cell content FFPE samples obtained indefinite results by FISH test. The six patients with positive ALK protein expression received crizotinib therapy, and 5 paitents got treated effectively. For two ALK IHC positive cases, which were 4% neutral buffered formalin fixed for 72 h, the result of Ventana IHC ALK (D5F3) staining became weakened obviously and uneven. In 10 cases of samples, total RNA was extracted from FFPE cytologic sections and cytospin prepared slides, and the results of qRT-PCR test and ALK gene fusion showed good concordance. The standardized protocol recommended in this study expands the detection types and quantity of cytologic specimens for ALK protein expression and gene fusion and increased the detection rate. Ventana IHC ALK (D5F3) is a reliable method for detecting ALK protein expression in FFPE cell blocks. The pathologic quality control procedure prior to Ventana IHC ALK (D5F3) is crucial for the accuracy of testing the ALK gene status. When FFPE cell blocks could not be prepared or prepared unsuccessfully from the cytologic specimens, qRT-PCR may be an alternative option for the detection of ALK gene fusion.

Lack of ROS1 Gene Rearrangement in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor, and the prognosis remains poor. Rearrangement of ROS1 gene, which was shown to have an oncogenic potential, was previously discovered in GBM cell lines. In this pilot study, we aimed to identify the incidence of ROS1 rearrangement in GBM patient tissues to explore novel biomarkers for therapeutic strategy. Formalin-fixed and paraffin-embedded (FFPE) tissue sections from 109 patients with GBM were screened for ROS1 rearrangement by anti-ROS immunohistochemistry (IHC) and ROS1 break-apart fluorescent in situ hybridization (FISH) assays. O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation and Isocitrate dehydrogenase 1 (IDH1) mutation status were also assessed. All samples were interpreted by two experienced pathologists who were blinded to the clinical data. A total of 109 samples were collected and all samples were examined for ROS1 rearrangement by IHC and FISH assays, and none was found to harbor ROS1 rearrangement. MGMT gene methylation was found in 42 (39.2%) cases, and IDH1 mutation was found in 6 (5.5%) cases. In this study, ROS1 rearrangement was not identified in GBM patients, and thus it is difficult to classify ROS1 rearrangement as a novel molecular subset in GBM patients for now.

Clinicopathologic characteristics and therapeutic responses of Chinese patients with non-small cell lung cancer who harbor an anaplastic lymphoma kinase rearrangement.

IntroductionThe rearrangement of the anaplastic lymphoma kinase (ALK) gene accounts for approximately 1%–6% of lung adenocarcinoma cases and defines a molecular subgroup of tumors characterized by clinical sensitivity to ALK inhibitors such as crizotinib. This study aimed to identify the relationship between ALK rearrangement and the clinicopathologic characteristics of non-small cell lung cancer (NSCLC) and to analyze the therapeutic responses of crizotinib and conventional chemotherapy to ALK rearrangement in NSCLC patients.MethodsA total of 487 lung cancer patients who underwent testing for ALK rearrangement in our department were included in this study. ALK rearrangement was examined by using fluorescence in situ hybridization (FISH) assay.ResultsAmong the 487 patients, 44 (9.0%) were diagnosed with ALK rearrangement by using FISH assay. In 123 patients with adenocarcinoma who were non-smokers and of a young age (≤58 years old), the frequency of ALK rearrangement was 20.3% (25/123). Short overall survival (OS) was associated with non-adenocarcinoma tumor type (P = 0.006), poorly differentiated tumors (P = 0.001), advanced-stage tumors (P < 0.001), smoking history (P = 0.008), and wild-type epidermal growth factor receptor (EGFR) (P = 0.008). Moreover, patients with poorly differentiated and advanced-stage tumors had a shorter time to cancer progression compared with those with well differentiated (P = 0.023) and early-stage tumors (P = 0.001), respectively.ConclusionsALK-rearranged NSCLC tends to occur in younger individuals who are either non-smokers or light smokers with adenocarcinoma. Patients with ALK rearrangement might benefit from ALK inhibitor therapy.

Abstract 4911: Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolor FISH

Abstract Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others (“mutual exclusivity”). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridization (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data was also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirms that RTK/RAS components are mutually exclusively amplified in GC, and demonstrates the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient, before a definitive diagnosis can be attained. Citation Format: Kakoli Das, Bavani Gunasegaran, Iain Beehuat Tan, Niantao Deng, Kiat Hon Lim, Patrick Tan. Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolor FISH. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4911. doi:10.1158/1538-7445.AM2015-4911

Abstract 4927: Next-generation sequencing enables new approach to molecular cytogenetics

Abstract Background: Accurate characterization of chromosomal aberrations (cytogenetics) in hematologic malignancies is fundamental for proper disease diagnosis, prognosis and may inform treatment decisions. Current karyotyping and fluorescent in situ hybridization (FISH) clinical assays have important sensitivity and sample limitations and cannot detect many novel chromosomal abnormalities in cancer. We have developed a NGS-based targeted DNA and RNA sequencing assay (FoundationOne Heme)and analysis pipeline compatible with typical clinical specimens that can detect all types of variants including large chromosomal abnormalities at base-pair level. High accuracy for the detection of recurrent cytogenetic events was validated across 100 CLL and DLBCL specimens with known abnormalities Method: Adaptor-ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 405 blood cancer-related genes by DNA-seq, and 265 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 498x for DNA and ∼7M on-target unique pairs for RNA, to enable highly accurate detection of base substitutions, indels, copy number alterations and gene rearrangements. Cytogenetics calls were determined using a computational method which compares germline SNP allele frequencies to model expectations, taking into account tumor purity, ploidy, and local copy number. Result: Assay performance demonstrated highly accurate detection of known cytogenetic events, including large deletions, trisomy and translocations, in 61 CLL and 39 DLBCL specimens previously characterized with CLIA-certified karyotyping and FISH assays. In the CLL cohort, 40 of 41 (98%) known positives and 172 of 172 (100%) known negatives were successfully characterized, including deletion of chr11q, chr13q, chr17p and chr12 trisomy. No false positive was reported in any of the cytogenetic variants. In the DLBCL cohort, 35 of 39 (90%) known translocations involving IGH-MYC, IGH-BCL2 and IGH-BCL6 were detected. Accurate detection of these rearrangements is particularly difficult as they are not typical gene fusions and the genomic breakpoints are highly variable, distributed across upstream, genic and downstream of the targeted genes. In addition, deletion in chr17p including TP53 was detected from 37 of 165 DLBCL patients with large length distribution ranging from 500 kb to 79 Mb. Conclusion: Accurate detection of cytogenetic events at higher resolution than traditional cytogenetics and FISH techniques is possible using an optimized NGS-based assay and tailored algorithms. An improved understanding of the molecular details of chromosomal abnormalities, integrated with comprehensive profiling of other cancer relevant genomic alterations, may help the upfront characterization of these alterations and impact diagnosis, prognosis and therapeutic intervention. Citation Format: Jie He, Jeffrey Gardner, James X. Sun, Omar Abdel-Wahab, Andrew M. Intlekofer, Michelle K. Nahas, Jo-Anne Vergilio, Jeffery S. Ross, Roman Yelenskey, Ross Levine, Geoff Otto, Doron Lipson, Vincent Miller, Philip Stephens. Next-generation sequencing enables new approach to molecular cytogenetics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4927. doi:10.1158/1538-7445.AM2015-4927

Novel fluorescence insitu hybridization-based definition of bacille Calmette-Guérin (BCG) failure for use in enhancing recruitment into clinical trials of intravesical therapies.

To present a molecular definition of bacille Calmette-Guérin (BCG) failure that incorporates fluorescence in situ hybridization (FISH) testing to predict BCG failure before it becomes clinically evident, which can be used to enhance trial designs for patients with non-muscle-invasive bladder cancer. We used data from 143 patients who were followed prospectively for 2 years during intravesical BCG therapy, during which time FISH assays were collected and correlated to clinical outcomes. Of the 95 patients with no evidence of tumour at 3-month cystoscopy, 23 developed tumour recurrence and 17 developed disease progression by 2 years. Patients with a positive FISH test at both 6 weeks and 3 months were more likely to develop tumour recurrence (17/37 patients [46%] and 16/28 patients [57%], respectively) than patients with a negative FISH test (6/58 patients [10%] and 3/39 patients [8%], respectively; both P < 0.001). Using hazard ratios for recurrence with positive 6-week and 3-month FISH results, we constructed clinical trial scenarios whereby patients with a negative 3-month cystoscopy and positive FISH result could be considered to have 'molecular BCG failure' and could be enrolled in prospective, randomized clinical trials comparing BCG therapy (control) with an experimental intravesical therapy. Patients with positive early FISH and negative 3-month cystoscopy results can be considered to have molecular BCG failure based on their high rates of recurrence and progression. This definition is intended for use in designing clinical trials, thus potentially allowing continued use of BCG as an ethical comparator arm.

Evaluation of HER2/neu Status by Immunohistochemistry Using Computer-Based Image Analysis and Correlation With Gene Amplification by Fluorescence In Situ Hybridization Assay: A 10-Year Experience and Impact of Test Standardization on Concordance Rate.

The American Society of Clinical Oncology/College of American Pathologists proposed several recommendations for human epidermal growth factor receptor 2 (HER2) test standardization. One suggestion was that image analysis (IA) could be useful for scoring of HER2/neu immunohistochemistry. The utilization of IA in a real-world practice in a large cohort of cases has not been previously reported. To compare HER2/neu quantification by IA with gene amplification by fluorescence in situ hybridization (FISH); to determine sensitivity, specificity, and concordance rates with the FISH assay; and to determine association between HER2 status with estrogen receptor (ER), progesterone receptor (PR), and Ki-67 expression. We evaluated HER2 results performed by immunohistochemistry and FISH in conjunction with ER, PR, and Ki-67 in 3093 invasive breast cancer cases from 2002 to 2011. The overall concordance between immunohistochemistry and FISH was 87.3% (1768 of 2026). When analyzed by year, there was an improvement in the positive concordance rate from 49.4% (44 of 89) to 95.0% (57 of 60) (P < .001). The negative concordance rate was at least 95% with a median false-negative rate of 1.5%. In the FISH+ group, amplification ratio showed significant correlation with IA scores (P < .001). Positive versus negative HER2 status was associated with lower ER and PR levels (P < .001) and higher Ki-67 expression (P < .001). Scoring of HER2/neu by IA was associated with high false-positive rates before 2008. Improvement in concordance rate after 2008 may be due to proper tissue handling, improved HER2/neu scoring by IA, and assay standardization.

P16/CDKN2A FISH in Differentiation of Diffuse Malignant Peritoneal Mesothelioma From Mesothelial Hyperplasia and Epithelial Ovarian Cancer.

It can be difficult to differentiate diffuse malignant peritoneal mesothelioma (DMPM) from reactive mesothelial hyperplasia (RMH) or peritoneal dissemination of gynecologic malignancies, such as epithelial ovarian cancer (EOC), which cause a large amount of ascites. Detection of the homozygous deletion of p16/CDKN2A (p16) by fluorescence in situ hybridization (FISH) is an effective adjunct in the diagnosis of malignant pleural mesothelioma. The aim of this study was to investigate the ability of the p16 FISH assay to differentiate DMPM from RMH and EOC. p16 FISH was performed in 28 DMPMs (successful in 19), 30 RMHs, and 40 EOC cases. The cutoff values of p16 FISH were more than 10% for homozygous deletion and more than 40% for heterozygous deletion. According to the above criteria, nine (47.4%) of 19 successful DMPM cases were homozygous deletion positive, and three (15.8%) of 19 were heterozygous deletion positive, whereas all RMH cases were negative for the p16 deletion. In all four major histologic subtypes of EOC, neither p16 homozygous nor heterozygous deletions were detected. To differentiate DMPM from RMH or EOC, the sensitivity of the p16 homozygous deletion was 32% (9/28), and the specificity was 100%. Our study suggests that p16 FISH analysis is useful in differentiating DMPM from RMH and EOC when homozygous deletion is detected.

Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence in Situ Hybridization by Quantitative Real-Time PCR

Administration of drugs targeting HER2 (official symbol ERBB2) is an important component of therapy for breast cancer patients with HER2 amplification/overexpression as determined by in situ hybridization (ISH) and immunohistochemistry (IHC). In approximately 5% of breast cancers, ISH assays fail. In these cases, HER2 protein expression is evaluated by IHC alone that may yield false negatives/positives for poor-quality samples. Therefore, we developed a method that was based on quantitative real-time PCR applicable for DNA from formalin-fixed, paraffin-embedded tissue samples. Its limit of detection was determined with breast cancer cell lines and validated with 223 breast cancer patient samples. On the basis of comparisons with fluorescent ISH (FISH) and IHC data, the sensitivity of the new method was 94.2% and 95.1%, its specificity was 100% and 99.1%, and overall concordance between results obtained with the quantitative real-time PCR method and FISH/IHC was 97.6% for both methods. The quantitative real-time PCR method was then used to evaluate the HER2 status of 198 of 3696 breast cancer tissues that yielded indeterminate FISH results. The HER2 copy number was successfully determined in 69.2% of these indeterminate samples. Thus, the DNA-based technique appears to be a specific, sensitive method for determining HER2 copy numbers when the FISH assay fails, which may complement IHC tests.

Newly designed break-apart and ASPL-TFE3 dual-fusion FISH assay are useful in diagnosing Xp11.2 translocation renal cell carcinoma and ASPL-TFE3 renal cell carcinoma: a STARD-compliant article.

The diagnosis of Xp11.2 translocation renal cell carcinoma (tRCC), which relies on morphology and immunohistochemistry (IHC), is often either missed in the diagnosis or misdiagnosed. To improve the accuracy of diagnosis of Xp11.2 tRCC and ASPL-TFE3 renal cell carcinoma (RCC), we investigated newly designed fluorescence in situ hybridization (FISH) probes (diagnostic accuracy study).Based on the genetic characteristics of Xp11.2 tRCC and the ASPL-TFE3 RCC, a new break-apart TFE3 FISH probe and an ASPL-TFE3 dual-fusion FISH probe were designed and applied to 65 patients with RCC who were <45 years old or showed suspicious microscopic features of Xp11.2 tRCC in our hospital. To test the accuracy of the probes, we further performed reverse transcriptase–polymerase chain reaction (PCR) on 8 cases for which frozen tissues were available.Among the 65 cases diagnosed with RCC, TFE3 IHC was positive in 24 cases. Twenty-two cases were confirmed as Xp11.2 tRCC by break-apart TFE3 FISH, and 6 of these cases were further diagnosed as ASPL-TFE3 RCC by ASPL-TFE3 dual-fusion FISH detection. Importantly, reverse transcriptase–PCR showed concordant results with the results of FISH assay in the 8 available frozen cases.The break-apart and ASPL-TFE3 dual-fusion FISH assay can accurately detect the translocation of the TFE3 gene and ASPL-TFE3 fusion gene and can thus serve as a valid complementary method for diagnosing Xp11.2 tRCC and ASPL-TFE3 RCC.

Quantification of human epidermal growth factor receptor 2 immunohistochemistry using the Ventana Image Analysis System: correlation with gene amplification by fluorescence in situ hybridization: the importance of instrument validation for achieving high (>95%) concordance rate.

The use of computer-based image analysis for scoring human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) has gained a lot of interest recently. We investigated the performance of the Ventana Image Analysis System (VIAS) in HER2 quantification by IHC and its correlation with fluorescence in situ hybridization (FISH). We specifically compared the 3+ IHC results using the manufacturer's machine score cutoffs versus laboratory-defined cutoffs with the FISH assay. Using the manufacturer's 3+ cutoff (VIAS score; 2.51 to 3.5), 181/536 (33.7%) were scored 3+, and FISH was positive in 147/181 (81.2%), 2 (1.1%) were equivocal, and 32 (17.6%) were FISH (-). Using the laboratory-defined 3+ cutoff (VIAS score 3.5), 52 (28.7%) cases were downgraded to 2+, of which 29 (55.7%) were FISH (-), and 23 (44.2%) were FISH (+). With the revised cutoff, there were improvements in the concordance rate from 89.1% to 97.0% and in the positive predictive value from 82.1% to 97.6%. The false-positive rate for 3+ decreased from 9.0% to 0.8%. Six of 175 (3.4%) IHC (-) cases were FISH (+). Three cases with a VIAS score 3.5 showed polysomy of chromosome 17. In conclusion, the VIAS may be a valuable tool for assisting pathologists in HER2 scoring; however, the positive cutoff defined by the manufacturer is associated with a high false-positive rate. This study highlights the importance of instrument validation/calibration to reduce false-positive results.

MP39-13 DEVELOPMENT OF A NOVEL METHOD FOR DETECTING RENAL CELL CARCINOMA CIRCULATING TUMOR CELLS

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2015MP39-13 DEVELOPMENT OF A NOVEL METHOD FOR DETECTING RENAL CELL CARCINOMA CIRCULATING TUMOR CELLS Michael Gorin, Mark Ball, Darren Davis, Phillip Pierorazio, Hans Hammers, Kenneth Pienta, and Mohamad Allaf Michael GorinMichael Gorin More articles by this author , Mark BallMark Ball More articles by this author , Darren DavisDarren Davis More articles by this author , Phillip PierorazioPhillip Pierorazio More articles by this author , Hans HammersHans Hammers More articles by this author , Kenneth PientaKenneth Pienta More articles by this author , and Mohamad AllafMohamad Allaf More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.763AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The isolation of circulating tumor cells (CTCs) from patients with renal cell carcinoma (RCC) has been met with limited success. This is due to the fact that most available CTC isolation technologies rely on the positive selection of cells using the surface protein EpCAM, an epithelial marker which is expressed in a minority of RCCs. ApoStream (ApoCell Inc., Houston, TX) is a novel technology which utilizes dielectrophoresis and microfluidics for the antibody-free isolation of CTCs. In this study, we developed a novel method for detecting RCC CTCs using the ApoStream platform and fluorescence in situ hybridization (FISH) for loss of the VHL gene. This assay was then tested in three patients with metastatic clear cell RCC. METHODS The optimal operating frequency for enrichment of RCC CTCs was determined using fluorescently labeled 786-O cells spiked in peripheral blood mononuclear cells from healthy donors. In parallel, conditions were optimized for performing FISH for the VHL gene (ZytoLight VHL/centromere 3 probe, ZytoVision GmbH, Germany) on isolated cells. Following successful assay development, 7.5 mL of blood from each of three patients with metastatic clear cell RCC was enriched for CTCs using the ApoStream instrument. From each sample, 1400 isolated mononuclear cells were evaluated with the optimized FISH assay. A CTC was defined as any mononuclear cell demonstrating net loss of the VHL gene. RESULTS Five operating frequencies (25, 45, 65, 85, 105 Hz) were evaluated for the optimal isolation of 786-O cells from peripheral blood mononuclear cells. Among the tested frequencies, 85 Hz demonstrated the highest mean rate of cell recovery (65%). In clinical testing, two of three (66.7%) patients had detectable CTCs (3 and 5 CTCs each). Figure 1 includes representative images of cells isolated from these two patients. CONCLUSIONS Antibody-independent isolation with dielectrophoresis and subsequent FISH for the VHL gene is a promising novel method for CTC detection in patients with metastatic RCC. Future work aims to validate this assay in larger patient cohort. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e459 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Gorin More articles by this author Mark Ball More articles by this author Darren Davis More articles by this author Phillip Pierorazio More articles by this author Hans Hammers More articles by this author Kenneth Pienta More articles by this author Mohamad Allaf More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...